Defining the Genetic Causes of Antifungal Drug Resistance in Cryptococcus neoformans

The higher rates of both morbidity and mortality among immunocompromised people significantly increased the prevalence and severity of fungal infection, resulting in 1.5 million annual deaths globally. The fungal pathogens that represent 90% of systematic fungal infections are opportunistic yeasts and moulds, particularly species belonging to the genus Candida, Cryptococcus and Aspergillus. Cryptococcus neoformans, an encapsulated yeast, commonly causes serious meningoencephalitis and meningitis; these conditions are treated using fluconazole (FLC), the most common azole antifungal agent used today. However, the widespread usage of antifungal drugs reduces the drugs’ effectiveness and results in treatment failure due to the evolution of resistant strains. According to the latest report by the World Health Organization (WHO), which lists the globally recognised fungal priority pathogens (FPPs), C. neoformans is identified as one of the top primary threats to public health. In Australia, over 4,000 deaths were estimated due to serious fungal infections, and the mean annual incidence of cryptococcosis among HIV patients was about 25% yearly. This number significantly increased up to 90% in cryptococcal patients without HIV, including solid organ transplantation, cancer, or other immunocompromising diseases such as systemic inflammatory disease, diabetes, or chronic kidney or liver dysfunction. Azole resistance is the most serious issue, as it is mostly used in long-term monotherapy. The microevolution of azole resistance in C. neoformans has been moderately studied, but the underlying genetic mutations remain unclear. Therefore, this research project conducted a literature review (Chapter 1) to determine the knowledge gaps in the microevolution of C. neoformans antifungal drug resistance mechanisms. The study was designed to achieve multiple objectives, representing thesis chapters and investigating the contribution of mutations, genetic metabolic function and aneuploidy to the microevolution of antifungal resistance in C. neoformans. Chapter 2 focused on the microevolutionary emergence of azole antifungal drug resistance in C. neoformans, which identified the genetic determinants causing FLC resistance in C. neoformans msh2 resistant mutants. Whole genome sequencing data analysis revealed the mutational profile and genes carrying mutations. This study provided evidence that msh2, a mismatch repair pathway gene, has no role in transient and permanent aneuploidy but has a standard microevolutionary route to the emergence of antifungal resistance. This involved the accumulation of mutations in genes that alter stress signalling, cellular efflux, membrane trafficking, epigenetic modification, and aneuploidy. This complex pattern of microevolution highlighted the significant challenges posed by the diagnosis and treatment of drug-resistant fungal pathogens. The roles of these genes in the evolution of antifungal drug resistance were investigated further in Chapters 3 and 4. Chapter 3 investigated the molecular mechanisms underlying antifungal drug resistance in C. neoformans, which examined the genes mutated in resistant isolates shown by genome sequencing (in Chapter 2). The analysis revealed insight into the changes in the chromosomal and putative metabolic functions that occur in response to drug exposure. The findings revealed that fluconazole exposure led to global genomic changes and a rapid, transient aneuploid response in C. neoformans, occurring within 24 hours, which highlights the remarkable plasticity of its genome. The findings also showed that FLC caused permanent aneuploidy in C. neoformans, highlighting a higher prevalence of permanent aneuploidy in both heterogeneous wildtype and mutant populations; identified critical pathways associated with drug resistance, which could be targeted for the development of new drug treatments and improve efficacy. Further studies were conducted to investigate permanent chromosomal changes contributing to antifungal drug resistance in Chapter 4. Chapter 4 examined the microevolution of FLC resistance and the influence of pre-exposure on genes regulating cell division, DNA repair, cell wall integrity, osmotic tolerance, and telomere length. This study mainly focused on the genome plasticity of C. neoformans wildtype and mutants by evaluating acute 15-day evolution assay for 250 generations in the absence and presence of low FLC concentration in C. neoformans. The analysis revealed increased copy numbers of chromosomes 5 and 7, indicating the potential association of FLC resistance in C. neoformans. This suggests that selective amplification of chromosomal regions containing genes involved in relevant cellular processes may contribute to resistance. This study provided more insight into the role of CDC2801, CRK1, MRC1 and RAD53 genes in fluconazole resistance. These genes are members of the CMGC and CAMK protein kinases, which play a pivotal role in the regulation of cell cycle progression, proliferation, cell growth, checkpoint signalling and DNA damage response, and are associated with cancer development and progression. Chapter 5 highlighted the main contributions of this work and suggested future applications. Specifically, this study provided evidence that the whole genome is amplified in the presence of FLC rather than specific chromosome amplification. It also demonstrated that resistance to FLC can evolve independently of ERG11 mutations, the most frequently observed and reported mechanism. Instead, a common evolutionary route to the emergence of resistance was identified in msh2 mutants. This is the first study that characterised the polygenic emergence of resistance through point mutations in Cryptococcus. Hence, this study should contribute to the development of more targeted genetic therapy to eliminate the emergence of resistance strains.